Project Summary Anti-factor VIII (FVIII) alloantibodies (inhibitors), which can develop in patients with hemophilia A, limit the therapeutic options for these patients, and can increase morbidity and mortality. However, not all patients with severe hemophilia A develop inhibitors following FVIII exposure. The ability to predict inhibitor development a priori would allow prophylactic interventions to be directed toward at-risk patients. However, there is currently no method capable of predicting those patients who will develop inhibitors following FVIII exposure. Furthermore, even if individuals who develop inhibitors could be identified prior to FVIII exposure, unlike transplantation, no prophylactic strategy currently exists that can actively prevent inhibitor formation. The inability to predict or prevent inhibitor formation in at-risk patients largely stems from a fundamental lack of understanding regarding key pathways that initiate this process. Our long-term goal is to identify the mechanisms that initiate and then orchestrate inhibitor formation, in order to predict and then prevent the development of anti-FVIII alloantibodies in patients with hemophilia A. Our central hypothesis is that marginal zone (MZ) B cells represent a central node in the initiation and orchestration of immune responses to FVIII, and thus may be a viable immunological target for the development of novel strategies to not only predict, but also prevent inhibitor formation. Our hypothesis is formulated on the basis of our recent discovery that FVIII rapidly co-localizes with MZ B cells shortly after injection and removal of MZ B cells prevents inhibitor formation. As MZ B cells and marginal zone macrophages (MZM) possess the ability to work in concert to initially trap circulating antigen, followed by MZ B cell-mediated trafficking of antigen to the B cell follicle and direct activation of CD4 T cells, these preliminary results suggest that MZ B cells and MZM likely work in concert to initiate FVIII inhibitor formation. Furthermore, as MZ B cells possess a restricted and distinct repertoire of antibody specificities within a given individual, these data suggest that differences in the precursor frequency of FVIII specific MZ B cells within a given patient may predict the likelihood that an individual with hemophilia A will develop inhibitors following FVIII exposure. We will use a complementary approach of clinical and pre-clinical studies to weld observational data with mechanistic studies in order to define the role of MZM and MZ B cells in inhibitor formation by testing the following specific aims: Aim 1: Define the role of MZM and MZ B cells in orchestrating FVIII inhibitor formation. Aim 2: Define the ability of dominant anti-FVIII MZ B cell clones to predict inhibitor formation in patients with hemophilia A. We believe that successful completion of these aims not only possess the capacity to provide new mechanistic insight into key aspects of inhibitor formation, but may also establish an important framework to develop rational approaches designed to prophylactically predict and ultimately prevent inhibitor development in patients with hemophilia A.